An ?-secretase inhibitor differentially affects the formation of ?-amyloid peptides in cultured cell lines additional evidence for multiple processing pathways. The amyloid plaques that accumulate in the neuropil and cerebral blood vessels of Alzheimer disease victims conain an array of peptides, which are cleaved from the juxta-membrane and transmembrane domains of the ?-amyloid peptide presursor (APP). The enzyme that cleaves APP(695) at Met-597 to yield Asp-1 of A? has been denoted ?-secretase, whereas (-secretase cleaves APP(695) at either Val-636 or Ala-638 to yield the C-terminus of A? (1-40) or A? (1-42). The enzyme ?-secretrase cleaves APP(695) at Lys-612, near the middle of the A?-forming sequence. An ??secretase in CHO cells appears to be the zinc-metalloprotease TACE, a tumor necrosis factor-?converting enzyme. Both TACE and ?-secretase are stimulated by phorbol esters, and both are inhibited by certain peptide-hydroxamate derivatives such as TAPI. The PMA stimulated ?-secretase is virtually absent in fibroblasts from mice lacking the TACE gene, which confirms that TACE is essential for this activitiy. We sought to determine whether ?-secretase competes with ?-secretase in the processing of APP, that is, whether ?-secretase action can preclude A? formation. We chose to study APP processing in two non-transfected cell lines rat PC-12 pheochromocytoma cells, which exhibit some properties of neuronal cells, and human A-204 rhabdomyosarcoma cells, which have been found to form A? deposits. In PC-12 cells TAPI inhibited ?-secretase to the extent of about 50%, and the yields of A??(1-40) and A? (11-40) were elevated about 2-fold. In contrast, in A-204 cells TAPI inhibited A? formation by about 60%. Mass spectral analysis of the products showed that, in addition to the usual form of A?, A-204 produced a unique form, which resulted from ?-secretase cleavage 3 residues before Met-597. The cleavage that produces this form is strongly inhibited by TAPI. These results show that A? can be formed through different pathways, which may give unexpected responses to inhibitors, either lowering or raising the rate of A? secretion depending upon the cell-type. It appears likely that more than one ?-secretase and one ?-secretase exist in these cells, since saturating amounts of the inhibitor only lowered the rate of cleavage by about 50%. One ?-secretase and one ?-secretase are Zn-metalloproteases.